In the honey bee (Apis) sex is determined by the complementary sex
determining locus or csd (Beye et al. , 2003), located on linkage group 3 of the honey bee genome (Solignac et al. ,
2007). Diploid individuals that are heterozygous at
the csd are female, whereas individuals that are haploid, and therefore
hemizygous at the csd are male (Cook and Crozier, 1995). Rarely, a diploid individual will be homozygous at the
csd, usually due to inbreeding. These individuals develop as diploid males,
but are killed by workers at the first larval instar (Woyke, 1963). Diploid males can be reared artificially, but doing so is tedious (Polaczek
et al. , 2000).

Sometimes it is desirable to be able to determine the ploidy of honey bee eggs. For example,
it may be useful to determine if low egg viability is due to the presence of haploid eggs in
worker cells, or to other causes such as policing of worker-laid eggs or disease. Although
there are effective protocols for examining metaphase chromosomes from the gonads of adult
queens and drones (Hoshiba and Kusanagi, 1978; Hoshiba
and Okada, 1986) or from the brain ganglia of pre-pupae
(Stanimirovic et al. , 2005) we have been unable to find
a procedure suitable for examining metaphase chromosomes in eggs. Here we report a simple
protocol for determining the ploidy of honey bee eggs based on Imai et al. (1988), who provide elaborate details of the fixation
procedure.

To obtain biological material we caged a queen on an empty comb comprising worker and drone
sized cells overnight. We then collected 10 eggs from worker cells on three consecutive days
after oviposition and 12 three day-old eggs from drone cells. We cut freshly harvested eggs in
half and incubated them in a hypotonic colchicine solution (0.005% colchicine in 1% sodium
citrate solution) on a clean glass slide for 40 minutes. We then drained the solution off the
slide and saturated the tissue with freshly-prepared fixative solution I (60% 1:1
acetic-ethanol: glacial acetic acid 3 mL/ethanol (99.5%) 3 mL/distilled water 4 mL). We then
dissociated the tissue with two needles and quickly added two drops of fixative II (1:1
acetic-ethanol: glacial acetic acid 2 mL/ethanol (99.5%) 2 mL; freshly prepared) over the
spread, draining off the fixative I and blotting it from the edge of the slide using strips of
filter paper. Fixative III (100% glacial acetic acid) was then dripped over the preparation
while draining off the remaining fixative II, blotting it from the edge of the slide. We then
air dried the slides overnight and stained them the following day with freshly prepared Giemsa
solution (3% in Sorensen’s phosphate buffer pH 6.8) for 15 minutes at room temperature.

Slides were examined under light microscope and photographed using a Zeiss Axiophot
photomicroscope coupled with Olympus DP71 colour camera.

Preparations of day-old eggs did not reveal any cells suitable for cytogenetic analysis. Only
a few nuclei could be seen around the egg’s micropile and these showed features of prophase
cells such as loose chromatin and an absence of nucleoli (Fig. 1A). Two- day old eggs showed typical interphase cells with compact chromatin and
the presence of nucleoli (Fig. 1B). Only the preparations
from three-day old eggs showed cells in metaphase, 6 per slide on average. These cells showed
the expected chromosome number for A. mellifera, n = 16 or 2n = 32 (Fig.
1C and D).

Although our technique yields a relatively small number of metaphase cells per slide, it
allows a rapid assessment of ploidy level in eggs. The technique is potentially useful for the
detection of worker-laid (haploid) eggs in worker-cells and queen cells or determining if
individuals homozygous at multiple loci are diploid or haploid. Hybridization techniques such
as GISH or FISH with specific probes for known genes and subsequent counting of positive
hybridization spots could be applied in two-day old eggs, as preparations from eggs of this
age showed nuclei suitable for this purpose.

Acknowledgments

We thank Malcolm Ricketts for his help with photo-microscopy. RMB is supported by a
post-doctoral Fellowship (Conselho Nacional de Desenvolvimento Científico e Tecnológico -
CNPq) and Endeavour Awards Research fellowship.